PROJECT SUMMARY The overall objectives of this NIH K08 Career Mentored Award are to identify and investigate new therapeutic targets for epithelial injury due to cigarette smoke in chronic obstructive lung disease (COPD), and to facilitate the development of essential skills that will allow the candidate to become an effective and independent physician-scientist. The candidate and her mentors have constructed a rigorous training plan that will provide the foundation for a strong and successful academic career. This proposal explores the core mechanisms of how adenine nucleotide translocase (ANT) is localized to the plasma membrane of ciliated airway epithelium and further how ANT is utilized for more advanced cellular functions, such as ciliary ATP regulation impacting airway hydration and ciliary beating. COPD is the 3rd leading cause of death in the US with cigarette smoke (CS) being the primary insult leading to disease. Despite ongoing mammalian research, no new biologic targets have been identified, and current therapies have limited effect on disease progression or mortality. I have leveraged the power of a genetically tractable model system, the amoebozoan Dictyostelium discoideum, as a genetic selection discovery tool for lung biology. I identified human ANT (ANT1 and ANT2 present in lung) as protective against CS-induced cell death in human bronchial epithelial cells. ANT is a mitochondrial ADP/ATP transporter that plays an active role in mitochondrial metabolism and ATP homeostasis. While exploring the localization of ANTs in human airway tissue, I made a fascinating observation that in addition to mitochondrial localization, human ANT1 and ANT2 localize to the plasma membrane of motile cilia in human airway and isolated primary airway cells (Normal Human Bronchial Epithelial cells, NHBE). GFP-tagged adenoviral ANT1 and ANT2 also go to the ciliary membrane (and mitochondria) in primary NHBEs and ANT1 and ANT2 are present in isolated human ciliary axonemes by western analysis. In addition, ANT2 specifically enhances airway surface hydration and preserves ciliary beat frequency in the context of CS. This early career proposal answers the following questions: How does human ANT1 and ANT2 localize to the plasma membrane of airway epithelium; how do plasma membrane versus mitochondrial ANTs regulate airway metabolism, airway hydration and ciliary function in ciliated airway epithelium; and what is the impact of ANT2 loss and gain of function on airway dysfunction and COPD development through mouse models of disease given its protective phenotype on airway epithelium. This will be accomplished using a combination of molecular tools, ANT isoform genetic modification, cell and tissue imaging, real-time analysis of cellular metabolism, assessment of airway hydration and ciliary function, and correlation with a mouse model of smoke exposure. Ultimately, the role of ANT in lung disease has yet to be described and therapeutic manipulation of its biology may allow us to drive cells to a healthier state. The core biology themes explored here will likely have broad applicability and impact other lung diseases.